This research program focuses on transfer RNA biosynthesis and subcellular trafficking. In addition to their essential role in protein synthesis, tRNAs are required for nutrient signaling, regulation of apoptosis, protein degradation, and priming retroviral reverse transcription. Although for decades it was thought that tRNA movement is unidirectional, nucleus to cytoplasm, we and others discovered that tRNAs move bi-directionally between the nucleus and the cytoplasm and that the dynamics are conserved between yeast and vertebrate cells. tRNA dynamics is comprised of 3 steps: "initial export" of tRNA from the nucleus to the cytoplasm, "retrograde" nuclear import of cytoplasmic tRNAs, and "re-export" of the imported tRNAs back to the cytoplasm. The mechanisms by which tRNAs transit between the nucleus and the cytoplasm are not completely understood but they require at least 3 members of the 2-importin family. However, a major player(s) still needs to be identified since cells are healthy if the only known nuclear exporter for intron-containing tRNAs is absent. On the other hand, there are proteins that affect the nuclear import and re-export steps by unknown mechanisms. Thus, Aim 1 seeks to discover missing gene products and to learn how the known proteins function in tRNA subcellular dynamics. The biological role(s) of tRNA bi-directional trafficking remains unknown;however, we showed that tRNAs accumulate in nuclei when damaged or when cells are deprived of nutrients. We propose that there is competition between the numerous tRNA processing steps that occur in the nucleus and tRNA nuclear export, sometimes resulting in erroneous delivery of aberrant tRNAs to the cytoplasm and that the tRNA retrograde process may have evolved, in part, to correct such errors. Since cytoplasmic tRNAs accumulate in the nucleus when cells are acutely deprived of nutrients, we propose that the retrograde process serves an additional function to regulate protein synthesis in response to nutrient availability. Thus, Aim 2 explores whether tRNA subcellular dynamics serve roles in tRNA quality control and/or regulation of translation. In addition to the proteins that deliver tRNAs to their correct subcellular destinations, tRNA biogenesis requires a complex set of ~80 independent gene products for post- transcriptional processing. Most of the processing activities are conserved throughout eukaryotes. Our data indicate that the conserved yeast pre-tRNA splicing endonuclease serves an additional essential novel cytoplasmic function(s) and Aim 3 probes the putative role(s). Discovery of a novel role(s) of the yeast enzyme may help explain how alterations of human tRNA splicing enzyme cause a fatal spinal muscular disease without causing defects in pre-tRNA splicing. Thus, the proposed research program impacts upon multiple facets of gene expression, response to environmental signals, and issues important to human health. PUBLIC HEALTH RELEVANCE: This research program focuses on transfer RNA biosynthesis and subcellular trafficking in eukaryotic cells. In addition to their essential role in protein synthesis, tRNAs are required for nutrient signaling, regulation of apoptosis, protein degradation, and priming retroviral reverse transcription and numerous diseases are associated with defects in tRNA biogenesis. Here we employ the yeast model system to test the hypothesis that the tRNA splicing machinery serves a novel role in addition to its known role in removing introns from precursor tRNAs, results of which may explain a human disorder;we also study the mechanisms of action and the function of the complicated tRNA intracellular dynamics, one goal of which is to test a new hypothesis regarding its function in tRNA quality control.